PeerJ: Neurosciencehttps://peerj.com/articles/index.atom?journal=peerj&subject=2150Neuroscience articles published in PeerJHermit crab response to a visual threat is sensitive to looming cueshttps://peerj.com/articles/40582017-11-302017-11-30Talya ShragaiXiaoge PingCameron ArakakiDennis GarlickDaniel T. BlumsteinAaron P. Blaisdell
Prior work in our lab has shown that an expanding image on a computer screen elicits a hiding response in the Caribbean terrestrial hermit crab (Coenobita clypeatus). We conducted two experiments to identify what properties of the expanding stimulus contribute to its effectiveness as a visual threat. First we found that an expanding geometric star evoked a strong hiding response while a contracting or full-sized stationary star did not. A second experiment revealed that the more quickly the stimulus expanded the shorter the latency to hide. These findings suggest that the anti-predator response to looming stimulus relies heavily on visual cues relating to the manner of approach. The simulated visual threat on a computer screen captures key features of a real looming object that elicits hiding behavior in crabs in the wild.

Prior work in our lab has shown that an expanding image on a computer screen elicits a hiding response in the Caribbean terrestrial hermit crab (Coenobita clypeatus). We conducted two experiments to identify what properties of the expanding stimulus contribute to its effectiveness as a visual threat. First we found that an expanding geometric star evoked a strong hiding response while a contracting or full-sized stationary star did not. A second experiment revealed that the more quickly the stimulus expanded the shorter the latency to hide. These findings suggest that the anti-predator response to looming stimulus relies heavily on visual cues relating to the manner of approach. The simulated visual threat on a computer screen captures key features of a real looming object that elicits hiding behavior in crabs in the wild.

Transcriptomic profiling of mTOR and ryanodine receptor signaling molecules in developing zebrafish in the absence and presence of PCB 95https://peerj.com/articles/41062017-11-292017-11-29Daniel F. FrankGalen W. MillerRichard E. ConnonJuergen GeistPamela J. Lein
The mechanistic target of rapamycin (mTOR) and ryanodine receptor (RyR) signaling pathways regulate fundamental processes of neurodevelopment, and genetic mutations within these pathways have been linked to neurodevelopmental disorders. While previous studies have established that these signaling molecules are expressed in developing zebrafish, a detailed characterization of the ontogenetic profile of these signaling molecules is lacking. Thus, we evaluated the spatiotemporal expression of key transcripts in mTOR and RyR signaling pathways in wildtype zebrafish at 24, 72 and 120 hours post fertilization (hpf). We further determined whether transcriptional profiles of a subset of genes in both pathways were altered by exposure to PCB 95 (2,2′,3,5′,6-pentachlorobiphenyl), a pervasive environmental contaminant known to cause developmental neurotoxicity in mammalian systems via RyR-dependent mechanisms. Quantitative PCR revealed that transcription generally increased across development. Genes in the signaling pathway upstream of the mTORC1 complex, and the RyR-paralogs, ryr2a and ryr3, were robustly upregulated, and in situ hybridization of ryr3 coincided with a transcriptional shift from muscle to neuronal tissue after 24 hpf. Static waterborne exposure to PCB 95 beginning at 6 hpf significantly altered transcription of genes in both pathways. These changes were concentration- and time-dependent, and included downregulation of rptor, a member of the mTORC1 complex, at both 72 and 120 hpf, and increased transcript levels of the RyR paralog ryr2b and downstream target of RyR signaling, Wingless-type 2ba (wnt2ba) at 72 hpf. The detailed transcriptomic profiling of key genes within these two signaling pathways provides a baseline for identifying other environmental factors that modify normal spatiotemporal expression patterns of mTOR and RyR signaling pathways in the developing zebrafish, as illustrated here for PCB 95.

The mechanistic target of rapamycin (mTOR) and ryanodine receptor (RyR) signaling pathways regulate fundamental processes of neurodevelopment, and genetic mutations within these pathways have been linked to neurodevelopmental disorders. While previous studies have established that these signaling molecules are expressed in developing zebrafish, a detailed characterization of the ontogenetic profile of these signaling molecules is lacking. Thus, we evaluated the spatiotemporal expression of key transcripts in mTOR and RyR signaling pathways in wildtype zebrafish at 24, 72 and 120 hours post fertilization (hpf). We further determined whether transcriptional profiles of a subset of genes in both pathways were altered by exposure to PCB 95 (2,2′,3,5′,6-pentachlorobiphenyl), a pervasive environmental contaminant known to cause developmental neurotoxicity in mammalian systems via RyR-dependent mechanisms. Quantitative PCR revealed that transcription generally increased across development. Genes in the signaling pathway upstream of the mTORC1 complex, and the RyR-paralogs, ryr2a and ryr3, were robustly upregulated, and in situ hybridization of ryr3 coincided with a transcriptional shift from muscle to neuronal tissue after 24 hpf. Static waterborne exposure to PCB 95 beginning at 6 hpf significantly altered transcription of genes in both pathways. These changes were concentration- and time-dependent, and included downregulation of rptor, a member of the mTORC1 complex, at both 72 and 120 hpf, and increased transcript levels of the RyR paralog ryr2b and downstream target of RyR signaling, Wingless-type 2ba (wnt2ba) at 72 hpf. The detailed transcriptomic profiling of key genes within these two signaling pathways provides a baseline for identifying other environmental factors that modify normal spatiotemporal expression patterns of mTOR and RyR signaling pathways in the developing zebrafish, as illustrated here for PCB 95.

The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter functionhttps://peerj.com/articles/40932017-11-272017-11-27Mason PosnerKelly L. MurrayMatthew S. McDonaldHayden EighingerBrandon AndrewAmy DrossmanZachary HaleyJustin NussbaumLarry L. DavidKirsten J. Lampi
Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish α-crystallin promoter function, how it compares to that of mammals, or whether mammalian α-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of α-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish α-crystallins. Our data showed that mouse and zebrafish αA-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a smaller percentage of embryos. Mouse αB-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region increased lens and notochord GFP expression while decreasing muscle expression, suggesting that the influence of mouse promoter control regions carries over into zebrafish embryos. The two paralogous zebrafish αB-crystallin promoters produced subtly different expression profiles, with the aBa promoter driving expression equally in notochord and skeletal muscle while the αBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish αA increased between 1 and 2 days post fertilization (dpf), αBa increased between 4 and 5 dpf, but αBb remained at baseline levels through 5 dpf. Parallel reaction monitoring (PRM) mass spectrometry was used to detect αA, aBa, and αBb peptides in digests of zebrafish embryos. In whole embryos, αA-crystallin was first detected by 2 dpf, peaked in abundance by 4–5 dpf, and was localized to the eye. αBa was detected in whole embryo at nearly constant levels from 1–6 dpf, was also localized primarily to the eye, and its abundance in extraocular tissues decreased from 4–7 dpf. In contrast, due to its low abundance, no αBb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian α-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved. An ontogenetic shift in zebrafish aBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.

Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish α-crystallin promoter function, how it compares to that of mammals, or whether mammalian α-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of α-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish α-crystallins. Our data showed that mouse and zebrafish αA-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a smaller percentage of embryos. Mouse αB-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region increased lens and notochord GFP expression while decreasing muscle expression, suggesting that the influence of mouse promoter control regions carries over into zebrafish embryos. The two paralogous zebrafish αB-crystallin promoters produced subtly different expression profiles, with the aBa promoter driving expression equally in notochord and skeletal muscle while the αBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish αA increased between 1 and 2 days post fertilization (dpf), αBa increased between 4 and 5 dpf, but αBb remained at baseline levels through 5 dpf. Parallel reaction monitoring (PRM) mass spectrometry was used to detect αA, aBa, and αBb peptides in digests of zebrafish embryos. In whole embryos, αA-crystallin was first detected by 2 dpf, peaked in abundance by 4–5 dpf, and was localized to the eye. αBa was detected in whole embryo at nearly constant levels from 1–6 dpf, was also localized primarily to the eye, and its abundance in extraocular tissues decreased from 4–7 dpf. In contrast, due to its low abundance, no αBb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian α-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved. An ontogenetic shift in zebrafish aBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.

Major depressive disorder (MDD) is highly prevalent, recurrent, and associated with functional impairment, morbidity, and mortality. Herein, we aimed to identify disruptions in functional connectomics among subjects with MDD by using resting-state functional magnetic resonance imaging (rs-fMRI). Sixteen subjects with MDD and thirty health controls completed resting-state fMRI scans and clinical assessments (e.g., Hamilton Depression Rating Scale (HAMD) and Hospital Anxiety and Depression Scale (HADS)). We found higher amplitude of low frequency fluctuations (ALFF) bilaterally in the hippocampus and amygdala among MDD subjects when compared to healthy controls. Using graph theoretical analysis, we found decreased clustering coefficient, local efficiency, and transitivity in the MDD patients. Our findings suggest a potential biomarker for differentiating individuals with MDD from individuals without MDD.

Adult zebra finches rehearse highly variable song patterns during sleephttps://peerj.com/articles/40522017-11-162017-11-16Brent K. YoungGabriel B. MindlinEzequiel ArneodoFranz Goller
Brain activity during sleep is fairly ubiquitous and the best studied possible function is a role in memory consolidation, including motor memory. One suggested mechanism of how neural activity effects these benefits is through reactivation of neurons in patterns resembling those of the preceding experience. The specific patterns of motor activation replayed during sleep are largely unknown for any system. Brain areas devoted to song production in the songbird brain exhibit spontaneous song-like activity during sleep, but single cell neural recordings did not permit detection of the specific song patterns. We have now discovered that this sleep activation can be detected in the muscles of the vocal organ, thus providing a unique window into song-related brain activity at night. We show that male zebra finches (Taeniopygia guttata) frequently exhibit spontaneous song-like activity during the night, but that the fictive song patterns are highly variable and uncoordinated compared to the highly stereotyped day-time song production. This substantial variability is not consistent with the idea that night-time activity replays day-time experiences for consolidation. Although the function of this frequent activation is unknown, it may represent a mechanism for exploring motor space or serve to generate internal error signals that help maintain the high stereotypy of day-time song. In any case, the described activity supports the emerging insight that brain activity during sleep may serve a variety of functions.

Brain activity during sleep is fairly ubiquitous and the best studied possible function is a role in memory consolidation, including motor memory. One suggested mechanism of how neural activity effects these benefits is through reactivation of neurons in patterns resembling those of the preceding experience. The specific patterns of motor activation replayed during sleep are largely unknown for any system. Brain areas devoted to song production in the songbird brain exhibit spontaneous song-like activity during sleep, but single cell neural recordings did not permit detection of the specific song patterns. We have now discovered that this sleep activation can be detected in the muscles of the vocal organ, thus providing a unique window into song-related brain activity at night. We show that male zebra finches (Taeniopygia guttata) frequently exhibit spontaneous song-like activity during the night, but that the fictive song patterns are highly variable and uncoordinated compared to the highly stereotyped day-time song production. This substantial variability is not consistent with the idea that night-time activity replays day-time experiences for consolidation. Although the function of this frequent activation is unknown, it may represent a mechanism for exploring motor space or serve to generate internal error signals that help maintain the high stereotypy of day-time song. In any case, the described activity supports the emerging insight that brain activity during sleep may serve a variety of functions.

Assessing Mongolian gerbil emotional behavior: effects of two shock intensities and response-independent shocks during an extended inhibitory-avoidance taskhttps://peerj.com/articles/40092017-11-132017-11-13Camilo Hurtado-ParradoCamilo González-LeónMónica A. Arias-HigueraAngelo CardonaLucia G. MedinaLaura García-MuñozChristian SánchezJulián CifuentesJuan Carlos ForiguaAndrea OrtizCesar A. Acevedo-TrianaJavier L. Rico
Despite step-down inhibitory avoidance procedures that have been widely implemented in rats and mice to study learning and emotion phenomena, performance of other species in these tasks has received less attention. The case of the Mongolian gerbil is of relevance considering the discrepancies in the parameters of the step-down protocols implemented, especially the wide range of foot-shock intensities (i.e., 0.4–4.0 mA), and the lack of information on long-term performance, extinction effects, and behavioral patterning during these tasks. Experiment 1 aimed to (a) characterize gerbils’ acquisition, extinction, and steady-state performance during a multisession (i.e., extended) step-down protocol adapted for implementation in a commercially-available behavioral package (Video Fear Conditioning System—MED Associates Fairfax, VT, USA), and (b) compare gerbils’ performance in this task with two shock intensities – 0.5 vs. 1.0 mA—considered in the low-to-mid range. Results indicated that the 1.0 mA protocol produced more reliable and clear evidence of avoidance learning, extinction, and reacquisition in terms of increments in freezing and on-platform time as well as suppression of platform descent. Experiment 2 aimed to (a) assess whether an alternate protocol consisting of a random delivery of foot shocks could replicate the effects of Experiment 1 and (b) characterize gerbils’ exploratory behavior during the step-down task (jumping, digging, rearing, and probing). Random shocks did not reproduce the effects observed with the first protocol. The data also indicated that a change from random to response-dependent shocks affects (a) the length of each visit to the platform, but not the frequency of platform descends or freezing time, and (b) the patterns of exploratory behavior, namely, suppression of digging and rearing, as well as increments in probing and jumping. Overall, the study demonstrated the feasibility of the extended step-down protocol for studying steady performance, extinction, and reacquisition of avoidance behavior in gerbils, which could be easily implemented in a commercially available system. The observation that 1.0 mA shocks produced a clear and consistent avoidance behavior suggests that implementation of higher intensities is unnecessary for reproducing aversive-conditioning effects in this species. The observed patterning of freezing, platform descents, and exploratory responses produced by the change from random to periodic shocks may relate to the active defensive system of the gerbil. Of special interest is the probing behavior, which could be interpreted as risk assessment and has not been reported in other rodent species exposed to step-down and similar tasks.

Despite step-down inhibitory avoidance procedures that have been widely implemented in rats and mice to study learning and emotion phenomena, performance of other species in these tasks has received less attention. The case of the Mongolian gerbil is of relevance considering the discrepancies in the parameters of the step-down protocols implemented, especially the wide range of foot-shock intensities (i.e., 0.4–4.0 mA), and the lack of information on long-term performance, extinction effects, and behavioral patterning during these tasks. Experiment 1 aimed to (a) characterize gerbils’ acquisition, extinction, and steady-state performance during a multisession (i.e., extended) step-down protocol adapted for implementation in a commercially-available behavioral package (Video Fear Conditioning System—MED Associates Fairfax, VT, USA), and (b) compare gerbils’ performance in this task with two shock intensities – 0.5 vs. 1.0 mA—considered in the low-to-mid range. Results indicated that the 1.0 mA protocol produced more reliable and clear evidence of avoidance learning, extinction, and reacquisition in terms of increments in freezing and on-platform time as well as suppression of platform descent. Experiment 2 aimed to (a) assess whether an alternate protocol consisting of a random delivery of foot shocks could replicate the effects of Experiment 1 and (b) characterize gerbils’ exploratory behavior during the step-down task (jumping, digging, rearing, and probing). Random shocks did not reproduce the effects observed with the first protocol. The data also indicated that a change from random to response-dependent shocks affects (a) the length of each visit to the platform, but not the frequency of platform descends or freezing time, and (b) the patterns of exploratory behavior, namely, suppression of digging and rearing, as well as increments in probing and jumping. Overall, the study demonstrated the feasibility of the extended step-down protocol for studying steady performance, extinction, and reacquisition of avoidance behavior in gerbils, which could be easily implemented in a commercially available system. The observation that 1.0 mA shocks produced a clear and consistent avoidance behavior suggests that implementation of higher intensities is unnecessary for reproducing aversive-conditioning effects in this species. The observed patterning of freezing, platform descents, and exploratory responses produced by the change from random to periodic shocks may relate to the active defensive system of the gerbil. Of special interest is the probing behavior, which could be interpreted as risk assessment and has not been reported in other rodent species exposed to step-down and similar tasks.

Character drawing style in cartoons on empathy induction: an eye-tracking and EEG studyhttps://peerj.com/articles/39882017-11-082017-11-08Yong-il LeeYeojeong ChoiJaeseung Jeong
In its most basic form, empathy refers to the ability to understand another person’s feelings and emotions, representing an essential component of human social interaction. Owing to an increase in the use of mass media, which is used to distribute high levels of empathy-inducing content, media plays a key role in individual and social empathy induction. We investigated empathy induction in cartoons using eye movement, EEG and behavioral measures to explore whether empathy factors correlate with character drawing styles. Two different types of empathy-inducing cartoons that consisted of three stages and had the same story plot were used. One had an iconic style, while the other was realistic style. Fifty participants were divided into two groups corresponding to the individual cartoon drawing styles and were presented with only one type of drawing style. We found that there were no significant differences of empathy factors between iconic and realistic style. However, the Induced Empathy Score (IES) had a close relationship with subsequent attentional processing (total fixation length for gaze duration). Furthermore, iconic style suppressed the fronto-central area more than realistic style in the gamma power band. These results suggest that iconic cartoons have the advantage of abstraction during empathy induction, because the iconic cartoons induced the same level of empathy as realistic cartoons while using the same story plot (top-down process), even though lesser time and effort were required by the cartoon artist to draw them. This also means that the top-down process (story plot) is more important than the bottom-up process (drawing style) in empathy induction when viewing cartoons

In its most basic form, empathy refers to the ability to understand another person’s feelings and emotions, representing an essential component of human social interaction. Owing to an increase in the use of mass media, which is used to distribute high levels of empathy-inducing content, media plays a key role in individual and social empathy induction. We investigated empathy induction in cartoons using eye movement, EEG and behavioral measures to explore whether empathy factors correlate with character drawing styles. Two different types of empathy-inducing cartoons that consisted of three stages and had the same story plot were used. One had an iconic style, while the other was realistic style. Fifty participants were divided into two groups corresponding to the individual cartoon drawing styles and were presented with only one type of drawing style. We found that there were no significant differences of empathy factors between iconic and realistic style. However, the Induced Empathy Score (IES) had a close relationship with subsequent attentional processing (total fixation length for gaze duration). Furthermore, iconic style suppressed the fronto-central area more than realistic style in the gamma power band. These results suggest that iconic cartoons have the advantage of abstraction during empathy induction, because the iconic cartoons induced the same level of empathy as realistic cartoons while using the same story plot (top-down process), even though lesser time and effort were required by the cartoon artist to draw them. This also means that the top-down process (story plot) is more important than the bottom-up process (drawing style) in empathy induction when viewing cartoons

EEG sensorimotor rhythms’ variation and functional connectivity measures during motor imagery: linear relations and classification approacheshttps://peerj.com/articles/39832017-11-082017-11-08Carlos A. Stefano FilhoRomis AttuxGabriela Castellano
Hands motor imagery (MI) has been reported to alter synchronization patterns amongst neurons, yielding variations in the mu and beta bands’ power spectral density (PSD) of the electroencephalography (EEG) signal. These alterations have been used in the field of brain-computer interfaces (BCI), in an attempt to assign distinct MI tasks to commands of such a system. Recent studies have highlighted that information may be missing if knowledge about brain functional connectivity is not considered. In this work, we modeled the brain as a graph in which each EEG electrode represents a node. Our goal was to understand if there exists any linear correlation between variations in the synchronization patterns—that is, variations in the PSD of mu and beta bands—induced by MI and alterations in the corresponding functional networks. Moreover, we (1) explored the feasibility of using functional connectivity parameters as features for a classifier in the context of an MI-BCI; (2) investigated three different types of feature selection (FS) techniques; and (3) compared our approach to a more traditional method using the signal PSD as classifier inputs. Ten healthy subjects participated in this study. We observed significant correlations (p < 0.05) with values ranging from 0.4 to 0.9 between PSD variations and functional network alterations for some electrodes, prominently in the beta band. The PSD method performed better for data classification, with mean accuracies of (90 ± 8)% and (87 ± 7)% for the mu and beta band, respectively, versus (83 ± 8)% and (83 ± 7)% for the same bands for the graph method. Moreover, the number of features for the graph method was considerably larger. However, results for both methods were relatively close, and even overlapped when the uncertainties of the accuracy rates were considered. Further investigation regarding a careful exploration of other graph metrics may provide better alternatives.

Hands motor imagery (MI) has been reported to alter synchronization patterns amongst neurons, yielding variations in the mu and beta bands’ power spectral density (PSD) of the electroencephalography (EEG) signal. These alterations have been used in the field of brain-computer interfaces (BCI), in an attempt to assign distinct MI tasks to commands of such a system. Recent studies have highlighted that information may be missing if knowledge about brain functional connectivity is not considered. In this work, we modeled the brain as a graph in which each EEG electrode represents a node. Our goal was to understand if there exists any linear correlation between variations in the synchronization patterns—that is, variations in the PSD of mu and beta bands—induced by MI and alterations in the corresponding functional networks. Moreover, we (1) explored the feasibility of using functional connectivity parameters as features for a classifier in the context of an MI-BCI; (2) investigated three different types of feature selection (FS) techniques; and (3) compared our approach to a more traditional method using the signal PSD as classifier inputs. Ten healthy subjects participated in this study. We observed significant correlations (p < 0.05) with values ranging from 0.4 to 0.9 between PSD variations and functional network alterations for some electrodes, prominently in the beta band. The PSD method performed better for data classification, with mean accuracies of (90 ± 8)% and (87 ± 7)% for the mu and beta band, respectively, versus (83 ± 8)% and (83 ± 7)% for the same bands for the graph method. Moreover, the number of features for the graph method was considerably larger. However, results for both methods were relatively close, and even overlapped when the uncertainties of the accuracy rates were considered. Further investigation regarding a careful exploration of other graph metrics may provide better alternatives.

A novel animal model for neuroinflammation and white matter degenerationhttps://peerj.com/articles/39052017-10-312017-10-31Baohu JiKerin HigaVirawudh SoontornniyomkijAtsushi MiyanoharaXianjin Zhou
Small interference RNA has been widely used to suppress gene expression. Three different short hairpin RNAs (shRNAs) against dopamine D1 receptor (Drd1), driven by mouse U6 promoter in self-complementary AAV8 vector (scAAV8), were used to silence mouse striatal Drd1 expression. Transduction of mouse striatum with all three scAAV8-D1shRNA viruses, but not the control scAAV8 virus, causes extensive neuroinflammation, demyelination, and axon degeneration. RNA interference is known to be coupled to the innate immune system as a host cell defense against virus infection. Activation of the innate immune system may play a causal role in the development of neuroinflammation and white matter degeneration, providing a novel animal model for multiple sclerosis (MS) and other neuroinflammatory diseases.

Small interference RNA has been widely used to suppress gene expression. Three different short hairpin RNAs (shRNAs) against dopamine D1 receptor (Drd1), driven by mouse U6 promoter in self-complementary AAV8 vector (scAAV8), were used to silence mouse striatal Drd1 expression. Transduction of mouse striatum with all three scAAV8-D1shRNA viruses, but not the control scAAV8 virus, causes extensive neuroinflammation, demyelination, and axon degeneration. RNA interference is known to be coupled to the innate immune system as a host cell defense against virus infection. Activation of the innate immune system may play a causal role in the development of neuroinflammation and white matter degeneration, providing a novel animal model for multiple sclerosis (MS) and other neuroinflammatory diseases.

Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horseshttps://peerj.com/articles/39652017-10-272017-10-27Constanza Stefania MenesesHeine Yacob MüllerDaniel Eduardo HerzbergBenjamín UbertiHedie Almagro BustamanteMarianne Patricia Werner
The role of glial cells in pain modulation has recently gathered attention. The objective of this study was to determine healthy spinal microglia and astrocyte morphology and disposition in equine spinal cord dorsal horns using Iba-1 and GFAP/Cx-43 immunofluorescence labeling, respectively. Five adult horses without visible wounds or gait alterations were selected. Spinal cord segments were obtained post-mortem for immunohistochemical and immunocolocalization assays. Immunodetection of spinal cord dorsal horn astrocytes was done using a polyclonal goat antibody raised against Glial Fibrillary Acidic Protein (GFAP) and a polyclonal rabbit antibody against Connexin 43 (Cx-43). For immunodetection of spinal cord dorsal horn microglia, a polyclonal rabbit antibody against a synthetic peptide corresponding to the C-terminus of ionized calcium-binding adaptor molecule 1 (Iba-1) was used. Epifluorescence and confocal images were obtained for the morphological and organizational analysis. Evaluation of shape, area, cell diameter, cell process length and thickness was performed on dorsal horn microglia and astrocyte. Morphologically, an amoeboid spherical shape with a mean cell area of 92.4 + 34 µm2 (in lamina I, II and III) was found in horse microglial cells, located primarily in laminae I, II and III. Astrocyte primary stem branches (and cellular bodies to a much lesser extent) are mainly detected using GFAP. Thus, double GFAP/Cx-43 immunolabeling was needed in order to accurately characterize the morphology, dimension and cell density of astrocytes in horses. Horse and rodent astrocytes seem to have similar dimensions and localization. Horse astrocyte cells have an average diameter of 56 + 14 µm, with a main process length of 28 + 8 µm, and thickness of 1.4 + 0.3 µm, mainly situated in laminae I, II and III. Additionally, a close association between end-point astrocyte processes and microglial cell bodies was found. These results are the first characterization of cell morphology and organizational aspects of horse spinal glia. Iba-1 and GFAP/Cx-43 can successfully immune-label microglia and astrocytes respectively in horse spinal cords, and thus reveal cell morphology and corresponding distribution within the dorsal horn laminae of healthy horses. The conventional hyper-ramified shape that is normally visible in resting microglial cells was not found in horses. Instead, horse microglial cells had an amoeboid spherical shape. Horse protoplasmic astroglia is significantly smaller and structurally less complex than human astrocytes, with fewer main GFAP processes. Instead, horse astrocytes tend to be similar to those found in rodent’s model, with small somas and large cell processes. Microglia and astrocytes were found in the more superficial regions of the dorsal horn, similarly to that previously observed in humans and rodents. Further studies are needed to demonstrate the molecular mechanisms involved in the neuron-glia interaction in horses.

The role of glial cells in pain modulation has recently gathered attention. The objective of this study was to determine healthy spinal microglia and astrocyte morphology and disposition in equine spinal cord dorsal horns using Iba-1 and GFAP/Cx-43 immunofluorescence labeling, respectively. Five adult horses without visible wounds or gait alterations were selected. Spinal cord segments were obtained post-mortem for immunohistochemical and immunocolocalization assays. Immunodetection of spinal cord dorsal horn astrocytes was done using a polyclonal goat antibody raised against Glial Fibrillary Acidic Protein (GFAP) and a polyclonal rabbit antibody against Connexin 43 (Cx-43). For immunodetection of spinal cord dorsal horn microglia, a polyclonal rabbit antibody against a synthetic peptide corresponding to the C-terminus of ionized calcium-binding adaptor molecule 1 (Iba-1) was used. Epifluorescence and confocal images were obtained for the morphological and organizational analysis. Evaluation of shape, area, cell diameter, cell process length and thickness was performed on dorsal horn microglia and astrocyte. Morphologically, an amoeboid spherical shape with a mean cell area of 92.4 + 34 µm2 (in lamina I, II and III) was found in horse microglial cells, located primarily in laminae I, II and III. Astrocyte primary stem branches (and cellular bodies to a much lesser extent) are mainly detected using GFAP. Thus, double GFAP/Cx-43 immunolabeling was needed in order to accurately characterize the morphology, dimension and cell density of astrocytes in horses. Horse and rodent astrocytes seem to have similar dimensions and localization. Horse astrocyte cells have an average diameter of 56 + 14 µm, with a main process length of 28 + 8 µm, and thickness of 1.4 + 0.3 µm, mainly situated in laminae I, II and III. Additionally, a close association between end-point astrocyte processes and microglial cell bodies was found. These results are the first characterization of cell morphology and organizational aspects of horse spinal glia. Iba-1 and GFAP/Cx-43 can successfully immune-label microglia and astrocytes respectively in horse spinal cords, and thus reveal cell morphology and corresponding distribution within the dorsal horn laminae of healthy horses. The conventional hyper-ramified shape that is normally visible in resting microglial cells was not found in horses. Instead, horse microglial cells had an amoeboid spherical shape. Horse protoplasmic astroglia is significantly smaller and structurally less complex than human astrocytes, with fewer main GFAP processes. Instead, horse astrocytes tend to be similar to those found in rodent’s model, with small somas and large cell processes. Microglia and astrocytes were found in the more superficial regions of the dorsal horn, similarly to that previously observed in humans and rodents. Further studies are needed to demonstrate the molecular mechanisms involved in the neuron-glia interaction in horses.